1. Field of the Invention
The present invention relates to a multilayer analytical element, and more particularly to an analytical element appropriately employable for dry analysis of substance contained in a body fluid including substance contained therein in micro-quantity.
2. Description of Prior Arts
As a method of analysis of substance contained in a liquid sample such as a body fluid, there has been heretofore utilized a wet analysis such as a method comprising a step of bringing said substance into contact with other substance (reagent) contained in a certain solvent so as to cause a detectable reaction such as a reaction directly or indirectly showing color-formation or color-change, and a step of detecting said reaction.
On the other hand, a dry analysis (i.e., dry analysis operation) developed for the purpose of simplifying the analytical procedures has been recently utilized. As a representative example of an analytical element utilized for the dry analysis, well known is a dry analytical element (also called dry analytical material or article) which is in the form of a sheet, film, strip or tape comprising basically a spreading layer for spreading a liquid sample containing substance to be detected (i.e., analyte), at least one reagent layer containing a reagent which directly or indirectly gives a certain detectable reaction in contact with the analyte, and a light-transmissive support. The analysis employing the multilayer analytical element is generally carried out by procedures of applying a liquid sample containing the analyte onto the liquid sample-spreading layer of the analytical element, subjecting the analytical element to incubation if necessary, detecting the resulting detectable reaction such as generation (formation) or change of color by a photometric means or the like, and determining amount of the analyte according to colorimetry.
There are known a variety of analytical systems utilizable in analysis of analyte employing the multilayer analytical element. Representative examples of the analytical systems are as follows.
(A) An analytical system for detecting and measuring a detectable reaction occurring between a reagent contained in the reagent layer and the analyte, such as a reaction showing generation or change of color. This analytical system is employable for analyses of analytes such as a variety of proteins, for instance, total protein, albumin and globulin, hemoglobin decomposition substances, for instance, free (i.e., non-conjugated or indirect) bilirubin and conjugated (i.e., direct) bilirubin.
(B) An analytical system employing a reagent layer containing at least two kinds of reagents, which comprises procedures of causing reaction between one of the reagents and the analyte to produce a reactive substance such as ammonia or hydrogen peroxide, bringing the reactive substance in contact with other reagent (e.g., dye precursor) to cause a detectable reaction such as generation or change of color, and detecting and measuring the reaction. This analytical system is employable for analysis in the case that the analyte is glucose; lipid such as cholesterol, trigliceride and free fatty acid; enzyme such as lactate dehydrogenase; urea and uric acid.
(C) An analytical system employing a reagent layer containing a non-diffusive reagent having a color-forming group, which comprises procedures of converting the reagent into a diffusive product carrying the color-forming group by reaction with analyte, separating the diffusive product from the unreacted non-diffusive reagent, causing a detectable reaction such as generation or change of color by bringing the diffusive product into contact with a chromogen substance such as coupler, and detecting and measuring the reaction. This analytical system is employable for analysis of polysaccharide hydrolase such as amylase.
(D) An analytical system employing a reagent layer containing a non-diffusive reagent having a detectable character (e.g., color), which comprises procedures of producing a diffusive product carrying the detectable character from the reagent through reaction with the analyte, separating the diffusive product from the unreacted non-diffusive reagent, and detecting and measuring the diffusive product. This analytical system is employable for analysis of polysaccharide hydrolase such as amylase.
As described above, the multilayer analytical element contains a reagent reactive to analyte and may further contain other reagent reactive to a product given by reaction between the analyte and the above-mentioned reagent. These reagents are selected appropriately to meet the purposes of analysis. Among these reagents, not a few kind of reagents easily deteriorate under certain surrounding conditions, such as light, heat, and pH condition. Further, some reagents need specific pH condition to show their reactivities, and hence these reagents should be used under specific surrounding pH condition.
As described above, one of the representative analytical systems includes detecting color generation or color change caused by the reaction between a color-forming regent and an analyte or a reaction product of the analyte with other reagent, said color-forming reagent and other reagent both being beforehand incorporated into the analytical element. As the color-forming reagent (i.e., dye precursor) for the analyses of various analytes according to the above-stated analytical system, a diazonium salt is generally employed because the diazonium salt undergoes very sensitive reaction and is easily available. However, the diazonium salt has a drawback in that the diazonium salt is decomposed upon exposure to light. It has been heretofore known that the diazonium salt is easily decomposable upon exposure to light but is relatively stable in a low pH range, namely, on the acidic side. Hence, it has been proposed that the diazonium salt be preserved in the presence of an acidic substance. Particularly, a carboxylic acid type polymer that can serve as a binder to form a diazonium salt-containing layer, providing an acidic condition, is generally employed in combination with the diazonium salt to prepare an analytical element containing a diazonium salt. However, the use of the carboxylic acid type polymer is not sufficiently effective to stabilize the diazonium salt.